The present invention relates generally to methods of treating patients suffering from conditions associated with or resulting from burn injuries by administration of bactericidal/permeability-increasing (BPI) protein products.
Burn trauma causes approximately two million injuries, 100,000 hospital admissions, and 10,000 deaths every year in the United States. [Marano et al., Surgery, Gynecology & Obstetrics, 170:32-38 (1990).] In the past, many victims did not survive the initial resuscitation period. Current survival rates and clinical outcomes have progressively improved with the advent of aggressive burn wound excision techniques, graft therapy, and superior intensive care facilities, along with a better understanding of postburn physiological factors and fluid requirements, but further improvements are needed. Currently, patient morbidity and mortality is associated largely with pulmonary infection and inflammatory lung disease, such as adult respiratory distress syndrome (ARDS). Pulmonary problems occur most often in patients with extensive cutaneous burns, patients with associated smoke inhalation injury, and the elderly.
Current therapy, for example, at a regional burn center, involves immediate treatment of the patient with burn injuries, focusing on maintaining the airway, breathing and circulation. Placement of an endotracheal tube and respiratory support using controlled ventilation may be necessary. Typically, intravenous lines are placed immediately to permit fluid resuscitation to treat burn shock, which occurs generally in patients with burns over 20% of the total body surface area (TBSA), and is exacerbated in the elderly and those with smoke inhalation injury to the lungs. Fluid resuscitation continues for approximately 24 hours following burn injury, the period of severe capillary permeability. Generally, during this time the patient is carefully monitored and fluid administration rates are adjusted to achieve optimal tissue and organ perfusion. Severe burns require the administration of colloids (protein solutions) since large amounts of plasma protein may leak into the interstitial tissue secondary to the capillary leak. Prophylactic antibiotics are generally not used for burns, since resistant organisms will rapidly be selected. Rather, serial samples from the patient (primarily the wounds and the lungs) are taken and cultured and treatment of suspected infections is based on the results of surveillance cultures and the identity of common microorganisms which may be endemic in the individual burn unit. Nutritional support is typically initiated as early as possible, preferably with enteral administration of high-protein diets. Intravenous (parenteral) alimentation is avoided in burn patients since it is associated with very high complication rates and does not support the gastrointestinal tract.
After initiation of such supportive treatment, current therapy involves debriding wounds of loose epithelium. Topical antimicrobial agents, usually silver sulfadiazine, are applied to the open wounds. Partial-thickness burns which are expected to heal are treated with daily debridements and continuation of topical antimicrobial agents. Burn wounds which are full-thickness or deep partial-thickness are considered for surgical excisional therapy, which is normally begun within several days, as soon as the patient is stable. Wound excision is followed by coverage of the open wounds with the patient's own skin (autograft) or with a temporary skin substitute, usually cadaver allograft, if the wounds are very extensive or if the patient is unstable. Temporary skin replacements are eventually replaced with autografts.
Acute burn injury initiates an early cytokine response in patients that involves tumor necrosis factor (TNF), interleukin-6 (IL-6) and interleukin-8 (IL-8). These cytokines are elevated in plasma and in local organs, including lung and skin. The cytokine responses in the lung and skin appear to be generated locally and do not originate from the systemic cytokine pool; local cytokine responses thus may play a greater role in local organ failure than the systemic response. Increased severity of burn injury or direct injury to the lung corresponds to increased systemic IL-8 levels, but not TNF or IL-6 levels. [Rodriguez et al., J. Trauma, 34:684-695 (1993).] TNF levels are detectable with greater frequency and at higher concentrations in burn patients with sepsis or a fatal outcome, but do not appear to correlate with the extent of burn injury. [Marano et al., supra; De Bandt et al., J. Trauma, 36:624-628 (1994).] One report has shown a correlation between elevated IL-6 levels postburn and the extent of burn injury. [De Bandt et al., supra.]
The inflammatory response and release of mediators that is associated with burn injury often results in vascular injury (increased permeability and hemorrhage) and tissue injury in the skin and in remote organs such as the lung. This vascular and tissue injury has been shown to be neutrophil-dependent. [Mulligan et al., Am. J. Pathol., 144:1008-1015 (1994).] Neutrophils respond to the systemic inflammation that initially follows major trauma by becoming primed and activated. Subsequent proinflammatory insults promote neutrophil deposition in tissues (leukosequestration) and release of proteases and oxygen metabolites from the sequestered neutrophils. The resulting vascular and tissue injury may lead to a self-sustaining cycle of neutrophil sequestration, additional vascular and tissue injury, and eventual end organ damage and failure. [Botha et al., Shock, 3:157-166 (1995).]
BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652 (1987)]. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in FIG. 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Gray et al. amino acid sequence is set out in SEQ ID NO: 1 hereto.
BPI is a strongly cationic protein. The N-terminal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of -3. [Elsbach and Weiss (1981), supra.] A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD has an amphipathic character, containing alternating hydrophobic and hydrophilic regions. This N-terminal fragment of human BPI possesses the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio. Chem., 262:14891-14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms. [Ooi et al., J. Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately 23 kD, referred to as "rBPI.sub.23, " has been produced by recombinant means and also retains anti-bacterial activity against gram-negative organisms. Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).
The bactericidal effect of BPI has been reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992). This reported target cell specificity was believed to be the result of the strong attraction of BPI for lipopolysaccharide (LPS), which is unique to the outer membrane (or envelope) of gram-negative organisms.
The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on LPS. LPS has been referred to as "endotoxin" because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, reported to be the most toxic and most biologically active component of LPS.
In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992), supra]. BPI is thought to act in two stages. The first is a sublethal stage that is characterized by immediate growth arrest, permeabilization of the outer membrane and selective activation of bacterial enzymes that hydrolyze phospholipids and peptidoglycans. Bacteria at this stage can be rescued by growth in serum albumin supplemented media [Mannion et al., J. Clin. Invest., 85:853-860 (1990)]. The second stage, defined by growth inhibition that cannot be reversed by serum albumin, occurs after prolonged exposure of the bacteria to BPI and is characterized by extensive physiologic and structural changes, including apparent damage to the inner cytoplasmic membrane.
Initial binding of BPI to LPS leads to organizational changes that probably result from binding to the anionic groups in the KDO region of LPS, which normally stabilize the outer membrane through binding of Mg.sup.++ and Ca.sup.++. Attachment of BPI to the outer membrane of gram-negative bacteria produces rapid permeabilization of the outer membrane to hydrophobic agents such as actinomycin D. Binding of BPI and subsequent gram-negative bacterial killing depends, at least in part, upon the LPS polysaccharide chain length, with long O-chain bearing, "smooth" organisms being more resistant to BPI bactericidal effects than short O-chain bearing, "rough" organisms [Weiss et al., J. Clin. Invest. 65:619-628 (1980)]. This first stage of BPI action, permeabilization of the gram-negative outer envelope, is reversible upon dissociation of the BPI, a process requiring the presence of divalent cations and synthesis of new LPS [Weiss et al., J. Immunol. 132: 3109-3115 (1984)]. Loss of gram-negative bacterial viability, however, is not reversed by processes which restore the envelope integrity, suggesting that the bactericidal action is mediated by additional lesions induced in the target organism and which may be situated at the cytoplasmic membrane (Mannion et al., J. Clin. Invest. 86: 631-641 (1990)). Specific investigation of this possibility has shown that on a molar basis BPI is at least as inhibitory of cytoplasmic membrane vesicle function as polymyxin B (In't Veld et al., Infection and Immunity 56:1203-1208 (1988)) but the exact mechanism as well as the relevance of such vesicles to studies of intact organisms has not yet been elucidated.
Of interest to the background of the present invention are reports of interaction between bacterial endotoxin and BPI protein products in various in vitro and non-human in vivo assay systems. As one example, Leach et al., Keystone Symposia "Recognition of Endotoxin in Biologic Systems", Lake Tahoe, Calif., Mar. 1-7, 1992 (Abstract) reported that rBPI.sub.23 (as described in Gazzano-Santoro et al., supra) prevented lethal endotoxemia in actinomycin D-sensitized CD-1 mice challenged with E. coli 011:B4 LPS. In additional studies Kohn et al., J. Infectious Diseases, 168:1307-1310 (1993) demonstrated that rBPI.sub.23 not only protected actinomycin-D sensitized mice in a dose-dependent manner from the lethal effects of LPS challenge but also attenuated the LPS-induced elevation of TNF and IL-1 in serum. Ammons et al., [Circulatory Shock, 41:176-184 (1993)] demonstrated in a rat endotoxemia model that rBPI.sub.23 produced a dose-dependent inhibition of hemodynamic changes associated with endotoxemia. Kelly et al., Surgery, 114:140-146 (1993) showed that rBPI.sub.23 conferred significantly greater protection from death than an antiendotoxin monoclonal antibody (E5) in mice inoculated intratracheally with a lethal dose of E. coli. Kung, et al., International Conference on Endotoxin IV, Amsterdam, Netherlands, Aug. 17-20, 1993 (Abstract 022) and in Bacterial Endotoxins: Basic Science to Anti-Sepsis Strategies, Wiley-Liss, N.Y., pages 255-263 (1994) disclosed the efficacy of rBPI.sub.23 in several animal models including live bacterial challenge and endotoxemia models. Von der Mohlen et al., 34th Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, Fla. Oct. 4-7, 1994 (Abstract M3), disclosed that rBPI.sub.23 administration alleviated serological, hematological and hemodynamic effects of endotoxin administration, including blunting the leukocyte response and reducing neutrophil activation.
M. N. Marra and R. W. Scott and co-workers have addressed endotoxin interactions with BPI protein products in U.S. Pat. Nos. 5,089,274 and 5,171,739, in published PCT Application WO 92/03535 and in Marra et al., J. Immunol., 144:662-665 (1990), Marra et al., J. Immunol., 148:532-537 (1992), and Marra et al., Critical Care Med., 22(4):559-565 (1994). In vitro and non-human in vivo experimental procedures reported in these documents include positive assessments of the ability of BPI-containing granulocyte extracts, highly purified granulocytic BPI and recombinant BPI to inhibit endotoxin stimulation of cultures of human adherent mononuclear cells to produce tumor necrosis factor .alpha. (TNF) when endotoxin is pre-incubated with the BPI product. Pre-incubation of endotoxin with BPI protein products was also shown to diminish the capacity of endotoxin to stimulate (upregulate) neutrophil cell surface expression of receptors for the complement system components C3b and C3bi in vitro. The experimental studies reported in these documents included in vivo assessments of endotoxin interaction with BPI protein products in test subject mice and rats.
Also of interest to the background of the present invention is a report showing a direct relationship between the extent of burn injury and gut permeability in rats, with additive increases in gut permeability seen in association with Pseudomonas aeruginosa infections in the burn wound. See Ryan et al., Arch. Surg., 129:325-328 (1994). Also of interest to the present invention is the report that BPI protein products reduce the adverse physiological effects, including cardiac and hemodynamic alterations, associated with intestinal ischemia/reperfusion injury in rats. See Ammons et al., Annual Meeting of Professional Scientific Research Scientists, Experimental Biology 94.sup..TM., Anaheim, Calif., Abstracts 1-3391, Part I, Ischemic Injury, 753 (Apr. 24-28, 1994).
Of further interest to the present invention is the report that granulocyte and macrophage proliferation is suppressed in burned and infected mice and appears to be related in part to endotoxin-stimulated production of prostaglandin mediators. [Gamelli et al., J. Trauma, 37:339-346 (1994).]
There continues to exist a need in the art for new methods and materials for treatment of burn injuries. Products and methods responsive to this need would ideally involve substantially non-toxic compounds available in large quantities by means of synthetic or recombinant methods. Ideal compounds would prevent or reduce the number and severity of complications associated with or resulting from burn injuries, or enhance the effect of other concurrently administered therapeutic agents, such as antibiotics or anti-fungal agents.